Schwann cell differentiation is thus a multistage process, the progression from one stage to the next being driven by dynamic alterations in underlying genetic programs. We hypothesize that microRNAs, being important developmental regulators in other systems, will be critical for SC developmental transitions. To evaluate the function of microRNAs in Schwann cell (SC) development we have conditionally ablated the microRNA processing enzyme, dicer. SC lacking dicer are stalled during differentiation and do not produce myelin, phenocopying models of congenital hypomyelination. Here in Specific Aim1, we will perform a detailed ultrastructural and molecular characterization of these SC lacking dicer. Preliminary analyses suggest that these SC are arrested at the promyelinating-myelinating transition, maintain the expression of Sox2, and lack the SC master regulator gene Egr2 (Krox20). To prove that loss of Egr2 is the underlying reason for dysmyelination, we will determine if forced expression of Egr2 can circumvent the phenotype of SC lacking dicer. Finally, we will generate a mosaic deletion of dicer, to verify that the changes observed in the dicer cKO are cell autonomous. In Specific Aim 2, we will perform microRNA profiling in developmental and pathological scenarios. We will identify key microRNAs that are dynamically expressed and then bioinformatically determine their candidate targets. We will then verify microRNA-target interactions using luciferase reporter assays in heterologous cells, argonaute co-immunoprecipitation, and overexpression in SC-neuron coculture systems. Finally we will determine whether some key microRNAs are themselves activated by Egr2, testing the hypothesis that Egr2 represses antecedent gene programs in SC with the help of microRNAs. Together these studies will be important in understanding the role of microRNAs in development. Further, since the processes underlying SC differentiation are considered a mirror image of SC dedifferentiation, these studies will not only shed light on developmental transitions but also on converse transitions that occur in peripheral myelin disorders and nerve injury. PUBLIC HEALTH RELEVANCE: This grant focuses on the role of microRNAs in the Schwann cell lineage. The experiments proposed will impact our understanding of both Schwann cell differentiation during development, as well as dedifferentiation in nerve injury and demyelinating diseases.